This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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1 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 Bioresource Technology 119 (2012) Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: Short Communication Enhancement of lipid accumulation in Scenedesmus obliquus by Optimizing CO 2 and Fe 3+ levels for biodiesel production Hanaa H. Abd El Baky a,, Gamal S. El-Baroty b, Abderrahim Bouaid c, Mercedes Martinez c, José Aracil c a Plant Biochemistry Department, National Research Centre, Dokki, Cairo, Egypt b Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt c Chemical Engineering Department, Faculty of Science Complutense University of Madrid, Madrid, Spain highlights " Scenedesmus obliquus is cultivated in nutrient medium supplemented with different levels either CO 2 and iron is established. " The maximum biomass and high total lipid are achieved at 12 % CO 2 and 20 mg/l iron. " The fatty acid composition of algal lipid is suitable for biodiesel. " Algal biodiesel is meet the specified of biodiesel standards. article info abstract Article history: Received 5 January 2012 Received in revised form 4 May 2012 Accepted 22 May 2012 Available online 1 June 2012 Keywords: Scenedesmus obliquus Microalgae biodiesel Fatty acid Microalgae oils Physiochemical properties of biodiesel The effects of cultivation of Scenedesmus obliquus in nutrient medium supplemented with 0.03%, 3, 9% and 12% CO 2 or mg L 1 of Fe 3+ on dry weight of biomass (DW), total lipid accumulation (TL contents) and total lipid productivity (TLP) were evaluated under indoor conditions. The accumulation of TL and TLP showed an increasing trend with increasing of CO 2 or Fe 3+ levels. In cultures with 12% CO 2 or 20 mg/l Fe 3+, maximum TL contents of 33.14% and 28.12%, respectively were obtained. These lipids displayed a fatty acid profile which is suitable for biodiesel production as the most abundant compounds were oleic ( %), palmitic ( %) and stearic ( % of total FAMEs) acids. The properties of biodiesel obtained from S. obliquus, were the same with those specification for biodiesel standards including ASTM D 6751 (American Society for Testing Material) and the European Standard En Thus, S. obliquus biomass could be used as suitable feedstock for biodiesel production. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Photosynthetic microalgae are considered as one of the most important renewable feedstocks for the production of biofuels, foods additives and health-care products (Demirbas and Demirbas 2011; Abd El-Baky and EI-Baroty 2011). Among the microalgal, Scenedesmus and Chlorella species have the most desirable features for efficient and economic combination of CO 2 fixation, wastewater treatment and lipid synthesis toward biofuel production (Tang et al., 2011). However, the economic feasibility of algae mass culture for biofuel production greatly depends on a high biomass productivity and valuable lipid yield (Liu et al., 2010). Lipids in microalgae could be modified by growth conditions such as nutrient limitation and salt stress (Ho et al., 2010; Abd El Baky et al., 2006). Therefore, the aim of the study is to investigate the effect of utilizing CO 2 as a carbon source as well as iron at various levels Corresponding author. address: abdelbakyh@hotmail.com (H.H. Abd El Baky). on Scenedesmus obliquus endemic in Egypt growth for produce biomass containing a high quantity of lipids content characterized for biodiesel production, which has not yet been investigated. 2. Materials and Methods 2.1. Algae culture S. obliquus was obtained from the Culture Collection of Algae at the University of Texas (Austin, TX, USA) from ten years ago. The cells were cultivated in 4 L Erlenmeyer flask with 3 L of N-9 medium (Borowitzka, 1988) at 25 ± 3.0 C under a light intensity of approximately 200 lem 2 s 1 provided by ten fluorescent lamp (Toshiba FL 40T8D/36). The initial ph of cultures was 7.0. The cultures were aerated with different flow rates of CO 2 mixed with ambient air to prepare CO 2 levels of 0.3%, 3.0%, 9.0% and 12%, and supplemented with 2.5 Fe 3+ mg/l as iron source. For the experiments on the effects of Fe ion concentrations, S. obliquus was cultivated in N-9 medium supplemented with Fe 3+ at 0.0, 2.5, 5, 10 and /$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.

3 430 H.H. Abd El Baky et al. / Bioresource Technology 119 (2012) mg/l (FeCl 3 ), and supplied with air stream containing 3% CO 2. The growth of S. obliquus culture was spectrophotometercally determined regularly every two days at 685 nm (OD 685 ). The dry weight (dw.) of algal biomass was determined gravimetrically. The calibration curve was prepared by drawing the relationship between OD 685 and dry weight Determination of lipid content After appropriate cell growth, the cells were harvested by centrifugation at 10,000g for 10 min and lyophilized. The total lipids were extracted with chloroform/methanol (2:1, v/v) and gravimetrically quantified (Bligh and Dyer, 1959) Biodiesel preparation Biodiesel from algal lipids was derived by acid catalyzed transesterification. Lipids were mixed with methanol with 1:56 molar ratio (weight ratio) and the reaction was carried out at 30 C for 4 h in the presence of sulfuric acid as catalyst with 1:1 weight ratio of catalyst to lipid Fatty acid analysis Fatty acids were converted into methyl esters (FAMEs) by direct transmethylation of lipid extracts with sulphuric acid in methanol (Christie, 2003). The FAMEs were analyzed by the Agilent Technologies 6890 N GC-system (USA) equipped with a flame ionization detector (FID) and a HP-5% Phenyl Methyl Silixane capillary column (30 m 0.32 mm i.d., 0.25 lm film thickness). Nitrogen was used as a carrier gas at a flow rate of 3.0 ml/min. The oven temperature was 70 C with a 2 min hold, raised to 230 C at8 C min 1 and held at 230 C for 20 min. Injector and detector temperatures were 250 and 280 C, respectively. FAMEs was identified by comparison its retention times with that of standard FAMEs mixture (Sigma, purity > 99.0% by GC) Measurement of physiochemical properties of biodiesel Acid value (AV), iodine value (IV) and peroxide value (PV) were estimated according to AOCS (1998) methods (Ca 5a-40), Cd 1 25 and Cd 8 53, respectively. The viscosity was measured with capillary viscosimeter in a constant temperature bath at 40 C. The oxidation rates (OR) and unsaturation degree (UD) were calculated based on GC analysis of FAME Statistical analysis All results are expressed as mean values ± S.D. Statistical differences between experimental groups were assessed by analysis of variance (ANOVA) using the COSTAT software package (Cohort Software, CA, USA). The mean values were compared with LSD test (P < 0.05). 3. Results and discussion 3.1. Effect of CO 2 concentrations on S. obliquus growth and total lipids The effect of CO 2 levels on the biomass (dw.), total lipid productivity and total lipid contents of S. obliquus cells are shown in Table 1. The values of these parameters were significantly different (P < 0.05) among all cultures, with various degrees. The maximum biomass of 1651 ± 262 mgdwl 1 was obtained at 9% CO 2. Several investigators reported that Chlorella sp., Nannochloropsis oculata, Dunaliella terticlecta and S. obliquus had optimal growth potential Table 1 Llipid production and growth rate of Scenedesmus obliquus cultivated in medum aerated with various CO 2 concentrations. and supplemented with different concentrations. CO 2 aeation % Biomass (cells dry weight mg 1 L) Total lipid productivity (mgl 1 d 1 ) in the range of 2 6% CO 2, and the growth was decreased by increasing CO 2 levels (Chiu et al., 2009; Tang et al., 2011; Fulke et al., 2010). However, S. obliquus SJTU-3 culture showed great abilities of CO 2 biofixation under the high CO 2 level (up to 20%) and produced the best growth potential at 10% CO 2. As shown in Table 1, the TL contents ( %,w/dw.) and TLP ( mgl 1 d 1 ) in S. obliquus cultures exhibited an increasing trend with the increase of CO 2 levels (0.3 12%). Therefore, the TL content was significantly correlated with the increase of TLP in S. obliquus cultures aerated with various CO 2 % levels Effect of CO 2 levels on fatty acid composition Lipid content % ± 210 a ± 263 a ± 262 b ± 105 a concentrations mg 1 L ± a ± a ± b ± c ± 0.85 b Each value represents the mean of three replicates and based on dry weight. All values are significant at (P < 0.5) (±): Standard Deviation. Gas chromatographic analysis of fatty acid methyl esters (FAME) derived from lipids of S. obliquus grown at 12% CO 2 (Table 2), indicate that the fatty acid composition would be acceptable to produce biodiesel and in accordance with that reported by other Scenedesmus species (Ho et al. 2010). In general, FAME from S. obliquus mainly contained the saturated fatty acids (SFA) and mono unsaturated fatty acids (MUFA) carbon chain length from Table 2 Fatty acid profile and criteria of lipid extracted from S. obliquus cultivated on nutrient media feeding with 12% CO 2 and 20 mg 1 L. Fatty acids a Relative content % b 12% CO 2 20 mg -1 L iron Lauric acid (C12:0) Myristic acid (C14:0) Palmitic acid (C16:0) Heptadecanoic acid (C17:0) Stearic acid (C18:0) Oletic acid (C18:1) Linoletic acid (C18:2) Linolenic acid (C18:3) Arachidic (C20:0) Lipid criteria Total saturated fatty acids Total monounsaturated fatty acid Total polyunsaturated fatty acids Total unsaturated fatty acids TU/TS DU RO U/TS: Total unsaturated / Total saturated DU: Degree of Unsaturated. TMSFA/ [Tdi = FA/100]+3 [T Tri = FA /100]. Rate of oxidation = [%UFA 1 = x1 / 100] + [%UFA 2 = x 12 /100]+ [%UFA 3 = x 25 /100]. (=): Number of double bond.. a Fatty acid was identified based on the total known fatty acids (Retention time, min). b The amount of the fatty acid was evaluted throw the peak area.

4 H.H. Abd El Baky et al. / Bioresource Technology 119 (2012) Cell numbers (106 ml-1) Culture agae (Days) 2.5 mg-1l 5 mg-1l 10 mg-1l 20 mg-1l Fig. 1. Effect of iron concentrations on growth of Scenedesmus obliquus. C16-C18 and lower amounts of polyunsaturated fatty acids (PU- FAs) (Table 2). The high proportion of SFA (57.45%) and MUFA (33.53%) in S. obliquus algae is considered as optimal composition for fuel quality standpoint. Ho et al. (2010) found that S. obliquus cultivated at 10% CO 2 had a relatively simple FA profile with C16:0 and C 18:1 as major FAs. In this study, S. obliquus contained C16:0 and C 18:1 as the major constituents, similar to those previously reported in Scenedesmus species (Ho et al., 2010). Generally, high CO 2 levels (5 20%) seem to be a proper condition for accumulation of high amounts of SFAs such as 16:0 and C14 especially in Scenedesmus species (Tang et al., 2011). The possible reason was that the increase in the CO 2 (>30%) level could lead to high CO 2 fixation and CO 2 consumption and evolved high O 2 level, that might affect the enzymatic of desaturation and elongation reactions (Vargas et al., 1998). The amounts of C16 and C18 species are similar to those found in Dunaliella, Chlorella, and Spirulina species used for biodiesel production (Tang et al., 2011) Effect of Fe 3+ concentrations on S. obliquus growth, total lipids, and fatty acid composition Maximum biomass production was achieved at 10 mg /L Fe 3+ (Fig. 1 and Table 1), whereas the highest TLP and TL contents in S. obliquus cultures were observed at 20 mgl 1 Fe 3+ (Table 3). The main MUFA in S. obliquus cultures supplemented with 20 mgl 1 Fe 3+ were C16:0 (25.12%), C18:0 (16.58%) and C18:1 (34.44%), which accounted for more than 76% of the TFAs (Table 2). This composition is suitable for biodiesel production (Miao and Wu, 2006; Miao et al., 2009). The SFA/MUFA content is similar to that obtained for S. obliquus (Ho et al., 2010; Yoo et al., 2010). The average PUFAs levels of S. obliquus (8.80% or 8.12%, of total FAME) are lower than those of other algae species, which may increase the oxidative stability of the biodiesel Characterization of biodiesel The biodiesel properties of S. obliquus lipids in general were comparable to those of biodiesel produced from vegetable oil tested according to the EN and B 100 specifications established by ASTM D requirements (Ramos et al., 2009). The biodiesel obtained from S. obliquus grown in medium supplemented with either 12% CO 2 or 20 mg Fe 3+ /L had lower acid value (0.42 and 0.40 mg KOH/g FAME) than that of the maximum value (<0.5 mg) recorded in the EN The values of density (kgm 3, at 15 C) and viscosity (in parentheses) (mm 2 /s, at 40 C) were (4.56) and (4.53) for biodiesel produced from S. obliquus grown in media containing either 12% CO 2 or 20 mg Fe 3+ /L. According to Tang et al. (2011) the algal lipids contained lower amounts of UFAs, which could be responsible for the lower values of viscosity and melting points. The relationship between FA composition in S. obliquus and the viscosity levels of biodiesel, indicates that C12 C18 fatty acids have viscosity values ranges of mm 2 /s (Moser, 2009). Thus, S. obliquus biodiesel may be expected to have a low viscosity level. Lower values of those parameters are desirable at low temperatures (Knothe, 2008). The IV of biodiesel of S. obliquus grown in media containing either 12% CO 2 or 20 mg Fe 3+ /L were 67 and 69 (mg I 2 /100 g), respectively. These values were much lower than the limit values (120 mgi 2 /100 g) of both European biodiesel and Spain Royal Decree (61/2006) (Ramos et al., 2009). A low IV for biodiesel FAME is necessary due to the fact that heating of USFAs results in polymerization (Mittelbach, 1996). Therefore, S. obliquus lipids, rich in SFA (57%) and low in PUFA (8.8%) may meet the European Standard for the level of IV. The degree of oxidation of unsaturated fatty compounds proceeds with different rates depending on the number and position of double bonds. In this study, S. obliquus lipids have low levels of C18:2 and C18:3 acids, which tend to give methyl ester fuels with high oxidative stability (Ramos et al., 2009). The peroxide value S. obliquus biodiesel was about zero. The values of OR and DU Table 3 Comparison of physiochemical properties of Scenedesmus obliquus biodiesel, diesel fuel and biodiesel standard. Properties Units Biodiesel of Sc. Oil grown at 12% CO 2 Biodiesel of Sc. Oil grown 20 mg 1 L Diesel a ASTM a Fuel Biodiesel Standard Density (Kg 1 L) Viscosity mm 2 S 1,at40 C Acid value mg KOH 1 g Max 0.5 Max 0.5 Peroxide number meq/kg ND ND Iodine value mg I 2 /100 g Max 120 a The data about diesel and ASTM diodiesel standard were taken from published literature as indicated in the text.

5 432 H.H. Abd El Baky et al. / Bioresource Technology 119 (2012) (in paraences) of biodiesel obtained from S. obliquus grow in media containing either 12% CO 2 or 20 mg Fe 3+ /L were 2.14 (0.52) and 2.20 (0.53) (Table 2). The low values of both OR and DU were associated with the high amounts of SFA or/and MUFA, which are refractory to auto-oxidation. The degree of unsaturation, double bond orientation, chain length has a high impact on the oil stability index (OSI) of fatty acid alkyl esters. Based on values of OSI (at 110 C, for 1 h) the stability of methyl esters of C12:0 18:0, C18:1, C18:2 and C18:3 were >40, 2.5, 1.0 and 0.2 h, respectively (Moser, 2009). By applying this observation, the biodiesel of S. obliquus exhibits superior oxidative stability. 4. Conclusion Lipid accumulation of 38% and 28% (w/dw) in S. obliquus cultures were obtained when grown in medium aerated with 12% CO 2 or supplemented with 10 mg Fe 3+ /L, respectively. The S. obliquus biodiesel contained high levels of C16:0 and C18:1 and low amounts of PUFA (C18:2, C18:3) methyl esters. The biodiesels displayed acceptable fuel properties including extremely low values of IV, AV, PV, density, viscosity and high oxidative stability. Thus, crude lipids obtained from S. obliquus could be a promising feedstock for biodiesel production. Acknowledgements The authors gratefully acknowledge financial support from the Egyptian and Spanish cooperation project (grant number A/028531/09). References Abd El-Baky, H. Hanaa, El-Baroty, G.S. (2011). Microalgae as a Source of Biofuel. In: Edie Morena (Ed.), J. Advances in Energy Research, vol. 5. Acosta, Nova Science Publishers, USA. pp Abd El Baky, H., Hanaa; El Baz, F.K., El-Baroty, G.S., Over-production of Lipid Rich in Linolenic Acid by Blue Green Alga Spirulina maxima and its Inhibitory Effect on Carcinoma Cells. Adv. Food Sci. 4, Bligh, E.G., Dyer, W.J., A rapid method of total extraction and purification. Can. J. Physiol. Pharmacol. 37, Borowitzka, M.A., Micro-Algal Biotechnology. Cambridge University Press, New York. Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., Lipid accumulation and CO 2 utilization of Nannochloropsis oculata in response to CO 2 aeration. Bioresour. Technol. 100, Christie, W.W., Phenyltridec-9-enoic and 15-phenylpentadec-9-enoic acids in Arum maculatum seed oil. Eur. J. Lipid Sci. Technol. 105, Demirbas, A., Demirbas, F., Importance of algae oil as a source of biodiesel. Energy Convers. Manage. 52 (1), Ho, S., Wen-Ming, C., Jo-Shu, C., Scenedesmus obliquus CNW-N as a potential candidate for CO 2 mitigation and biodiesel production. Bioresour. Technol. 101, Fulke, A.B., Mudliar, S.N., Raju, Y., Ajam, S., Srinivasan, N., Rishiram, R., Krishnamurthi, K., Bio-mitigation of CO 2, calcite formation and simultaneous biodiesel precursors production using Chlorella sp. Bioresour. Technol. 101, Knothe, G., Designer biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuel 22, Liu, J., Junchao, H., King, W.F., Yue, J., Yujuan, Z., Zheng, S., Feng, C., Production potential of Chlorella zofingienesis as a feedstock for biodiesel. Resour. Technol. (101), Miao, X., Wu, Q., Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97 (6), Miao, X.L., Li, R.X., Yao, H.Y., Effective acid-catalyzed transesterification for biodiesel production. Energy Converse Manage. 50, Mittelbach, M. (1996). The high flexibility of small scale biodiesel plants. Production of methyl esters in high quality using various feedstocks. in: Proceedings of the 2nd European Motor Biofuels Forum, Graz, Moser, B., Biodisel production, properties, and feedstocks. In Vitro Cell Dev. Biol. Plant. 45, Ramos, M.J., Fernández, C.M., Casas, Rodríguez, A., Pérez, A., Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour. Technol. 100, Tang, D., Han, W., Li, P., Miao, X., Zhong, J., CO 2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO 2 levels. Bioresour. Technology 102, Vargas, R.M., Ulf, S., Ricardo, S., Transesterification of vegetable oils: a review. J. Braz. Chem. Soc. 9 (1), Yoo, C., Jun, S.Y., Lee, J.Y., Ahn, C.Y., Oh, H.M., Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour. Technol. 101 (1), S71 S74.